Isotopic niche overlap between sympatric Australian snubfin and humpback dolphins

Abstract Ecological niche theory predicts the coexistence of closely related species is promoted by resource partitioning in space and time. Australian snubfin (Orcaella heinsohni) and humpback (Sousa sahulensis) dolphins live in sympatry throughout most of their range in northern Australian waters. We compared stable isotope ratios of carbon (δ13C) and nitrogen (δ15N) in their skin to investigate resource partitioning between these ecologically similar species. Skin samples were collected from live Australian snubfin (n = 31) and humpback dolphins (n = 23) along the east coast of Queensland in 2014–2015. Both species had similar δ13C and δ15N values and high (>50%) isotopic niche space overlap, suggesting that they feed at similar trophic levels, have substantial dietary overlap, and rely on similar basal food resources. Despite similarities, snubfin dolphins were more likely to have a larger δ15N value than humpback dolphins, indicating they may forage on a wider diversity of prey. Humpback dolphins were more likely to have a larger δ13C range suggesting they may forage on a wider range of habitats. Overall, results suggest that subtle differences in habitat use and prey selection are likely the principal resource partitioning mechanisms enabling the coexistence of Australian snubfin and humpback dolphins.

that promote coexistence and the trophic relationships among hightrophic level predators is critical to understand community structure and dynamics, and for ecosystem management and conservation.
Sympatric species with similar ecological requirements may compete for limited resources, which may lead to the exclusion of the less competitive species (Roughgarden, 1983). Coexisting species are thus expected to differ in ecological requirements to minimize niche overlap and avoid interspecific competition (Chesson, 2000;MacArthur & Levins, 1967). Niche differentiation can promote species coexistence; therefore, quantifying the degree of niche overlap among co-occurring species is an important tool for gaining insights into how closely related species coexist and the functional position and role they play within their environment (Broennimann et al., 2012;Geange et al., 2011;Lu et al., 1989).
Several species of delphinids coexist in sympatry (Bearzi, 2005;Parra, 2006;Syme et al., 2021). Delphinids eat a wide variety of prey, including fish, cephalopods, crustaceans, and even other marine mammals, and thus can act as apex-or mesopredators within marine and freshwater ecosystems (Kiszka et al., 2015). Given their high trophic position and relatively large body size, they can consume substantial amounts of prey biomass (Bearzi et al., 2010;Williams et al., 2004) and can have important effects on the overall trophic dynamics of marine ecosystems through direct predation and risk effects (Estes et al., 2016;Kiszka et al., 2015). However, relatively little is known about the ecological role of these dolphin communities, how they coexist, and their influences on the structure and function of marine ecosystems.
Australian snubfin (Orcaella heinsohni) and humpback dolphins (Sousa sahulensis), hereafter referred to as 'snubfin dolphin' and 'humpback dolphin', respectively, are primarily found in shallow (<30 m deep) tropical/subtropical coastal waters of the Sahul Shelf from the southern waters of New Guinea to, and across, northern Australian waters (Beasley et al., 2005;Jefferson & Rosenbaum, 2014). Both species co-occur throughout most of their range in northern Australian waters and are known to live in direct sympatry across several locations (Parra et al., 2002(Parra et al., , 2004. Ecologically, both species are relatively similar: both occur in small populations of typically fewer than 150 individuals, show a high degree of overlap in space use, and have similar patterns of habitat use and behavioral activities according to space and time, to the point that both species are recorded frequently in mixed species groups (Parra, 2005(Parra, , 2006Parra et al., , 2011. Thus, segregation into exclusive ranges in space and time, and difference in habitat use and behavior patterns, do not seem to fully explain their coexistence. Dietary niche partitioning is the primary way many carnivore species limit interspecific competition (Donadio & Buskirk, 2006) and a key mechanism regulating coexistence in marine mammals (Durante et al., 2021;Gibbs et al., 2011;Giménez et al., 2018). Slight differences in habitat preferences and diet appear to be some of the principal factors promoting the coexistence of snubfin and humpback dolphins (Parra, 2006;Parra & Jedensjö, 2014). Previous studies have shown that snubfin dolphins in northern Queensland preferred slightly shallower (1-2 m) waters than humpback dolphins (2-5 m), and favored seagrass beds more often than did humpback dolphins (Parra, 2006).
Stomach content analysis showed the diet of snubfin and humpback dolphins overlapped partially, particularly across the fish taxa consumed by both species (Parra & Jedensjö, 2014). However, humpback dolphins appeared to favor fish, while snubfin dolphin diet included a large amount of cephalopods (Parra & Jedensjö, 2014).
While stomach content analysis is a valuable tool in studies of diet composition, it can be biased due to inherent problems in the sampling regime and prey identification (Pierce & Boyle, 1991;Santos et al., 2001). Stomach contents can only be collected from dead stranded animals, which limits sampling opportunities (Barros et al., 2004;Matley et al., 2015), and stranded animals may have been engaged in abnormal feeding behavior before stranding due to illness (Owen et al., 2011); misrepresenting the actual diet of a healthy animal. Stomach contents are also biased toward hard parts such as otoliths and beaks, which are resistant to digestion, and may cause overestimation of the importance of particular prey such as cephalopods (Bowen & Iverson, 2013). In addition, erosion of hard parts may result in misidentification of prey species (Dunshea et al., 2013). These limitations prevent a clear understanding of dietary partitioning between sympatric species based on stomach content analyses alone.
Comparisons of carbon δ 13 C and nitrogen δ 15 N values among consumers (i.e., the isotopic niche) can provide a quantitative indication of an organism's trophic niche (Marshall et al., 2019;Newsome et al., 2007). Carbon isotope ratios can differ in a marine system due to temperature differences, surface-water CO 2 concentrations and differences in plankton biosynthesis or metabolism, and thus indicate likely carbon sources relating to feeding habitat (Ben-David & Flaherty, 2012;Kelly, 2000;Rubenstein & Hobson, 2004). Nitrogen isotope ratios can be used to estimate the consumer's trophic position given the well-established stepwise enrichment (3-4‰) of 15 N in the body tissue of organisms with increasing trophic level (Minagawa & Wada, 1984;Post, 2002).
To better understand the feeding ecology of snubfin and humpback dolphins, we investigated differences in their δ 13 C and δ 15 N values to assess isotopic niche width and overlap of niche space. We hypothesized that snubfin and humpback dolphins (1) have similar foraging habitats and trophic levels and that this would be reflected in comparable δ 13 C and δ 15 N values, (2) have similar trophic niches that would be reflected by a high degree of overlap of their isotopic niche spaces, and that (3) snubfin dolphins would have greater δ 15 N ranges, given they feed on a wider diversity of prey (fish and cephalopods) than humpback dolphins.

| Study area and sample collection
We collected skin samples of adult snubfin (n = 31) and humpback dolphins (n = 23) using a PAXARMS biopsy rifle (Krützen et al., 2002) during boat-based surveys in coastal waters of the Whitsundays and Capricorn-Curtis Coast region, east coast of Queensland ( Figure 1) between January 2014 and September 2015. Photos of each individual's dorsal fin were taken at the time of biopsy sampling for photo identification and to prevent re-sampling of individuals. Skin samples were transferred into liquid nitrogen prior to being stored at −80°C until stable isotope analysis at the Centre for Coastal Biogeochemistry Research, Southern Cross University.

| Sample preparation and stable isotope analysis
Preparation of skin samples followed standard protocols for stable isotope analysis . Approximately 10 mg of skin was cut from each sample using a stainless-steel scalpel sterilized with ethanol between cuts to prevent cross-contamination of samples. The skin pieces were then transferred into Eppendorf capsules and oven-dried at 60°C for 24 h to remove all moisture.
Once dried, samples were ground into a fine powder using a mortar and pestle (which were sterilized with acetone between samples).
Cetacean skin is known to have a high lipid content, which can lead to decreased δ 13 C values due to the 12 C enrichment in the lipids Lesage et al., 2010;Ryan et al., 2012). To minimize variance from lipid content all samples were lipid-extracted by adding 5 ml of 2:1 chloroform-methanol solution to the powdered samples, which were then vortexed for 30 s to ensure proper mixing . Lipid-extracted samples were then placed in a centrifuge for 5 min at 1000 rpm; the remaining solution was discarded and samples were again oven-dried at 60°C for 24 h to remove residual solvent. Depending on the amount of sample available after processing, aliquots of 0.05 to 0.9 mg of powdered sample were sealed in tin capsules. Samples were measured using a Thermo Fisher DELTA V plus isotope ratio mass spectrometer (IRMS). The IRMS was coupled to an elemental analyzer (Thermo Fisher Flash EA) via an interface (Thermo Fisher Conflo IV).
Isotopic ratios were transformed into parts per thousand (‰) using delta notation (δ): where δX is δ 13 C or δ 15 N, R sample is the ratio of light and heavier stable isotope in the sample, and R standard is the ratio of stable isotopes in the standard reference materials.

| Statistical analysis
We tested δ 15 N and δ 13 C data for each species for homogeneity of variance (non-parametric Levene's test) and normality (Shapiro-Wilk's test). Tests revealed homogeneity of variance for species, but assumptions of normality were not met for δ 13 C values for humpback dolphins (p = .04). We therefore used a one-sided randomization test with 10,000 permutations at 0.05 significance level to investigate differences in isotopic values between species. This test compares the difference of the mean δ 15 N and δ 13 C values per species with the difference obtained by randomly allocating the observed isotopic values among the two species (Manly, 2007).
We used six metrics, proposed by Layman et al. (2007), to compare the isotopic niches of Australian snubfin and humpback dolphins: 1. δ 15 N range, which is the difference between the highest and lowest δ 15 N values of each species. δ 15 N range provides information on the range of trophic levels at which each species has been feeding.
2. δ 13 C range, as a measure of the difference between the highest and lowest δ 13 C values of each species. δ 13 C range provides an estimate of the variability of trophic sources of each species.
3. Total area (TA), which is a measure of the total amount of niche space occupied by a species in ‰ 2 . TA was calculated from a convex hull drawn around the most extreme data points on an isotope δ 13 Cδ 15 N bi-plot. As TA is sensitive to differences in sample size, because the area can only increase as new data points are added, we used the corrected version of the standard ellipse area We bootstrapped all Layman metrics with replacement (n = 10,000, indicated with a subscript "boot") based on the smallest sample size in the data set (n = 23) to enable statistical comparison between dolphin species (Jackson et al., 2012;Manly, 2007).
To further assess niche widths and isotopic niche overlap between species, we followed a Bayesian approach using multivariate ellipsebased metrics . This method is particularly

While stable isotope analysis can provide vital information into
consumer-resource relationships, it is important to acknowledge that overlap in isotopic values of consumers does not necessarily indicate the same feeding habits or diet, as different prey species with similar isotopic values may produce similar δ 13 C and δ 15 N isotope values in their consumer's tissue (Phillips et al., 2014;Santos-Carvallo et al., 2015). Additionally, the resultant predator isotopic composition will also vary depending on the range of isotopic values and the relative proportions of ingested prey (Newsome et al., 2007;Phillips, 2001).
Furthermore, foraging strategies often vary with geographic location, sex, and age in delphinids (Gannon & Waples, 2004;Rossman et al., 2015), and these differences together with spatial and temporal variation in basal resource availability can affect dolphin diet and hence their carbon and nitrogen isotopic values (Ansmann et al., 2015;Browning, Cockcroft, et al., 2014;Peters et al., 2020) (Iverson et al., 2004) and compound-specific stable isotope analyses (Twining et al., 2020) could also allow for more fine-scale results. Despite these constraints, the results of this study are in line with our predictions and provide valuable insights into the trophic ecology of snubfin and humpback dolphins and a baseline for future studies.
Carbon isotope ratios in tissues of aquatic animals reflect the source of carbon at the base of the food chain and thus can be used to determine the habitat in which the predator has been feeding. (Kelly, 2000). In marine ecosystems, carbon isotope ratios tend to be more enriched in inshore/estuarine habitats than in offshore/ pelagic environments (France, 1995;Fry & Sherr, 1989). Snubfin and humpback dolphins had δ 13 C values in the range expected for marine predators living and foraging in nearshore systems (Clementz & Koch, 2001;Yves & Keith, 2007). Stomach content analyses of stranded and shark-net entangled dolphins revealed that both snubfin and humpback dolphins feed on a wide variety of fish and cephalopods associated with shallow coastal-estuarine environments (Parra & Jedensjö, 2014). These feeding habits are in accordance with behavioral observations indicating that snubfin and humpback dolphins often feed in shallow, coastal-estuarine habitats (Parra, 2006).
Interspecific differences in δ 15 N range are consistent with some degree of resource partitioning. The higher δ 15 N range observed in snubfin dolphins suggests they may feed on a slightly larger variety of prey resources than humpback dolphins. Stomach content analyses have shown that the main dietary difference between snubfin and humpback dolphins appears to be cephalopods, which were only found in large quantities in the stomachs of snubfin dolphins (Parra & Jedensjö, 2014). The cuttlefish and squid found in the stomachs of snubfin dolphins are abundant in shallow waters close to the coast (Jackson, 1991). In addition to cephalopods, snubfin dolphins also feed on schooling, bottom-dwelling, and pelagic fishes (Parra & Jedensjö, 2014). Thus, it is likely that the higher δ 15 N range in snubfin dolphins reflects a greater variation in the trophic level of their diet due to the consumption of cephalopods, as well as fish.
In contrast, bootstrapping indicated humpback dolphins were more likely to have a larger δ 13 C range than snubfin dolphins, suggesting they may use a slightly wider diversity of habitats.
Humpback dolphins are known to use a wide diversity of habitats associated with coastal waters, including dredged channels, inshore reefs, seagrass flats, and mangroves (Parra, 2006;Parra & Cagnazzi, 2016 Note: Isotopic means and ranges are given in ‰. Subscript "boot" indicates that the value (mean) has been generated via bootstrapping.
Abbreviations: CD, mean distance to centroid; MNND, mean nearest neighbor distance; SDNND, standard deviation of nearest neighbor distance; SEA, standard ellipse area; SEA B , Bayesian SEA; SEA C , standard ellipse area corrected for small sample size; TA, total area. TA B L E 1 Isotopic niche metrics (including the six Layman metrics) for Australian snubfin and humpback dolphins more than 50 km from the mainland coast in shallow shelf waters (i.e., <30 m deep) and near offshore islands off Queensland and Western Australia (Corkeron et al., 1997;Hanf et al., 2016;Parra et al., 2004;Raudino et al., 2018). Such sightings indicate that this species may use a wider range of different habitats, including intertidal areas around offshore islands. Alternatively, differences in δ 13 C range may reflect differences in basal resource availability across sampling locations in this study. The Capricorn Curtis region is characterized by a large inlet (Port Curtis), extended tidal- for humpback dolphins) was also consumed by the other (Parra & Jedensjö, 2014).
Ecological niche theory predicts that closely related species living in sympatry may compete for resources, unless there is resource partitioning through differences in dietary preferences, spatiotemporal habitat use patterns, and/or feeding behavior (MacArthur, 1968;Pianka, 1981). The differences in δ 13 C and δ 15 N range between snubfin and humpback dolphins found in this study suggest there is potentially some level of dietary and habitat partitioning, with snubfin dolphins foraging over a wider diversity of prey resources than humpback dolphins, and humpback dolphins utilizing a larger variety of habitats. Such differences in prey selection and habitat utilization most likely help minimize interspecific competition and have been observed among other sympatric communities of delphinids (Ansmann et al., 2015;Browning, Cockcroft, et al., 2014;Giménez, Cañadas, et al., 2017;Kiszka et al., 2011).
At the same time, the substantial overlap (>50%) in the mean core area (40%) of each species' isotopic niche space (SEA) suggests ecologically significant dietary overlap and potentially direct resource competition. Differences in dolphin feeding behavior may facilitate resource partitioning and reduce interspecific competition for shared prey resources such as fish. Humpback dolphins frequently forage behind trawlers, while snubfin dolphins have never been observed engaging in this behavior (Parra, 2006).
Alternatively, coastal-estuarine environments along the coast of Queensland are highly productive areas (Brodie et al., 2007), and as such, may provide abundant resources and promote snubfin and humpback dolphins' coexistence despite a great deal of overlap in their diet.
Prey diversity and abundance are key factors promoting the coexistence of upper-level predators as interspecific competition pressure tends to increase with decreasing prey availability and diversity (Holbrook & Schmitt, 1989;McArthur & Levins, 1967;Santos et al., 2019 (Kiszka et al., 2022), cumulative anthropogenic pressures on their prey need to be considered when planning future multi-species conservation.

| CON CLUS IONS
Our results confirm and strengthen results from earlier studies that suggested there is a high overlap in the diet and habitat use of snubfin and humpback dolphins (Parra, 2006;Parra & Jedensjö, 2014). We thank all the volunteers involved with the Capricorn Cetacean Project that assisted in obtaining samples and data in the field. We are grateful to Dr Matheus Carvalho de Carvalho for assistance with sample processing for isotopic analysis. KJP was supported by a Postdoc Grant from the University of Zurich.

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest in this study.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data associated with this manuscript have been deposited in Dryad at: https://doi.org/10.5061/dryad.95x69 p8n0.